The use of cellular immunotherapy against cancer has been thoroughly investigated since the introduction of lymphokine-activated killer (LAK) cells in the mid-1980s (Grimm E A. et al., 1982; Rosenberg S., 1985).
One of the most experimented approaches has been adoptive transfer of autologous or allogeneic cytotoxic effectors with tumor cell killing potential to trigger a graft-vs-tumor (GvT) effect. Among the various effector populations that have a potential anti-tumor effect, natural killer (NK) and NK-like T cells stand out with their high cytotoxic capacity (Sutlu T and Alici E., 2009).
NK and NK-like T cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs) and effector cell preparations such as LAK cells. Therefore methods involving current good manufacturing practice (cGMP)-compliant components that allow expansion of polyclonal NK cells and NK-like T cells in cell culture flasks using PBMCs from healthy donors (Carlens S. et al., 2001 and U.S. Ser. No. 10/242,788), as well as patients with B-cell chronic lymphocytic leukemia (Guven H. et al., 2003), and multiple myeloma (MM) (Alici E. et al., 2008) have been developed. These cells have been shown to exert specific cytotoxic activity against fresh human tumor cells in vitro and in experimental models of human tumors (Guimaraes F. et al., 2006) which opens up the possibility to be evaluated in clinical settings. However, the conventional flask-based culture is labor-intensive and cumbersome, thus limiting the cell number that can be handled practically. Previously disclosed protocols (e.g. Miller J S. et al., 1994; Pierson B A. et al., 1996; Luhm J. et al., 2002; Klingermann H G and Martinson J., 2004) directed to effector cell preparation also include steps such as NK precursor or CD56 separation prior to culture and the use of feeder cells or cGMP-incompatible components. These disadvantages render previous protocols suboptimal and unfeasible to support large clinical studies.
Furthermore, expansion of NK cells in cell culture flasks has the inherent risk of exposure to external agents and contamination. Although this risk is minimized in GMP laboratory environments, the use of closed automated systems is definitely preferred as long as it supplies sufficient amounts of cells.
Since hematopoietic cells are relatively sensitive to shear it is reasonable to assume that high shear processes are unsuitable for ex vivo expansion (Nielsen, 1999). Thus, stirred-tank bioreactors (Pierson. et al., 1996) or perfusion culture systems relying on external filters and high flow rate are unlikely to provide a high efficiency.
There are many promising approaches for the treatment of cancer with NK cell and NK-like T cell based immunotherapy. However, ex vivo expansion and activation of these effector cells under GMP compatible closed systems are crucial factors for facilitating frequent clinical use.
There have also been attempts by other investigators to expand and/or activate NK cells ex vivo and treatment options using purified/resting, short term or highly purified and long term activated NK cells are being investigated. These studies report NK cell infusions to be well tolerated and partially effective. Yet, the protocols used for effector cell preparation commonly include additional steps such as NK precursor or CD56 separation prior to culture and the use of feeder cells and/or cGMP-incompatible components. These disadvantages render such protocols suboptimal for GMP production and unfeasible for supporting large clinical studies.
A thorough evaluation of the above-mentioned reports shows a need for an automated method for optimized ex vivo expansion of NK cells. One problem with conventional flask-based cultures is related to scale, i.e. the cell number is limited due to the cumbersome handling of the flasks. Further, the risk of infections is quite high since the system is exposed to the environment each time the media are changed or the cells split. Further problems to be solved involve developing a method for the expansion of effector cells which is cost-effective, easy to handle and which includes well-defined cGMP quality components. Preferably the culture system is also free of animal products and feeder cells.